Efficacy of a Ceftazidime-Avibactam Co mbination in a...

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Efficacy of a Ceftazidime-Avibactam Combination in a Murine Septicemia 1

model caused by Enterobacteriaceae species producing AmpC or 2

Extended-Spectrum β-lactamases 3

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Premavathy Levasseura, Anne-Marie Girardb, Ludovic Lavalladec, Christine 5

Miossecd, John Pacee, Kenneth Colemanf 6

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Running Head: Ceftazidime-avibactam in vivo efficacy 8

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Corresponding author: P. Levasseur, Enghien Les Bains, France 10

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Novexel SA, Romainville, France 12

Present Address: aEnghien Les Bains, France; bInstitute De Recherche Servier, Croissy 13

Sur Seine, France; c Lantheus Medical Imaging, Saint-Laurent, Québec, Canada; 14

dVetoquinol, Paris, France; eStiefel Co., Research Triangle Park, North Carolina, USA; 15

fStow, MA, USA 16

AAC Accepts, published online ahead of print on 18 August 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.03579-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 17

Avibactam is a novel non-β-lactam β-lactamase inhibitor that has been shown in vitro to 18

inhibit class A, C and some class D β-lactamases. It is currently in phase 3 of clinical 19

development in combination with ceftazidime. In this study, the efficacy of ceftazidime-20

avibactam was evaluated in a murine septicemia model against five ceftazidime-21

susceptible (MICs 0.06 – 0.25 µg/ml) and 15 ceftazidime-resistant (MICs 64 - >128 22

µg/ml) species of Enterobacteriaceae, bearing either TEM, SHV, CTX-M extended-23

spectrum or AmpC β-lactamases. 24

In the first part of the study, ceftazidime-avibactam was administered at ratios of 4:1 and 25

8:1 (weight/weight) to evaluate the optimal ratio for efficacy. Against ceftazidime-26

susceptible isolates of Klebsiella pneumoniae and Escherichia coli, ceftazidime and 27

ceftazidime-avibactam demonstrated similar efficacy (unit dose ED50 of <1.5 – 9 mg/kg), 28

whereas, against ceftazidime-resistant β-lactamase-producing strains (ceftazidime unit 29

dose ED50 of >90 mg/kg), the addition of avibactam restored efficacy to ceftazidime (unit 30

dose ED50 dropped to <5-65 mg/kg). 31

In a subsequent study, eight isolates (two AmpC and six CTX-M producers) were 32

studied in the septicemia model. Ceftazidime-avibactam was administered at 4:1 33

weight/weight ratio and efficacy compared to 4:1 weight/weight ratios of either 34

piperacillin-tazobactam or cefotaxime-avibactam . Against the eight isolates, 35

ceftazidime-avibactam was the more effective combination, with unit dose ED50 values 36

ranging from 2-27 mg/kg compared to >90 mg/kg and 14->90 mg/kg for piperacillin-37

tazobactam or cefotaxime-avibactam, respectively. 38


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This study demonstrates that the potent in vitro activity observed with the ceftazidime-39

avibactam combination against ceftazidime-resistant Enterobacteriaceae bearing class 40

A and class C β-lactamases translated into good efficacy in the mouse septicemia 41

model. 42


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INTRODUCTION 43

Bacterial resistance plays a prominent role in determining treatment options and 44

currently represents a major public health issue. β-lactam antibiotics are active against 45

a wide range of bacterial pathogens and have low toxicity to humans, so globally 46

increasing levels of resistance to these agents are a particularly serious concern (1, 2). 47

In Gram-negative organisms, one of the most important mechanisms of resistance to β-48

lactams is the enzymatic cleavage of the β-lactam ring by β-lactamases (3). These 49

enzymes are grouped into four classes based on their amino acid sequences. Classes 50

A, C, and D β-lactamases contain a serine residue at the catalytic site, while class B 51

enzymes contain one or more zinc atoms (4). One very successful strategy to overcome 52

β-lactamase-mediated resistance is to combine the β-lactam antibiotic with a β-53

lactamase inhibitor, such as clavulanic acid, tazobactam or sulbactam. These currently 54

marketed β-lactamase inhibitors have a limited spectrum of clinical utility as their 55

inhibitory activity is confined, generally, to class A and a few class D β-lactamases. 56

Avibactam is the first of a new class of non-β-lactam β-lactamase inhibitors, 57

referred to as diazabicyclooctanes (5). It displays a broad spectrum of inhibitory activity 58

against both class A and class C β-lactamases, inactivating enzymes efficiently at low 59

IC50 values, with low turn-over numbers (6, 7). It has very little intrinsic antibacterial 60

activity, but efficiently protects β-lactams from hydrolysis by a wide variety of class A, 61

class C, and some class D- producing strains (8 -13), including extended-spectrum β-62

lactamases (ESBLs) (14), Klebsiella pneumoniae carbapenemase (KPC) (15, 16) and 63

OXA-48 producers (17). The β-lactamase landscape is changing radically, with KPC 64

carbapenemases and CTX-M type ESBLs now causing major resistance problems 65


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around the world (18, 19). Both in vitro and in vivo studies of combinations of oxyimino-66

cephalosporins with avibactam have been reported to overcome these resistances (20-67

22). While there is an extensive literature on the in vitro activities of ceftazidime-68

avibactam combinations, only a few studies on the in vivo efficacy of this combination 69

have been published against E. coli, K. pneumoniae, E. cloacae, C. freundii, and 70

Pseudomonas aeruginosa (20-27). 71

In this study, 20 isolates of Enterobacteriaceae were studied in a murine acute 72

lethal septicemia model, with most isolates producing ESBL and/or AmpC β-lactamases. 73

The objectives of the study were to: 74

(i) Evaluate the in vivo efficacy of ceftazidime with or without avibactam at two 75

different ratios of 4:1 and 8:1 and compare the relative efficacy of the 76

combination to that of commercially available amoxicillin-clavulanate 2:1 ratio and 77

piperacillin-tazobactam 8:1 ratio. 78

(ii) Evaluate the efficacy of ceftazidime, piperacillin, and cefotaxime with or without 79

avibactam and/or tazobactam at 4:1 ratio against AmpC or CTX-M producing E. 80

cloacae, E. coli and K. pneumoniae isolates. 81

Some of this work has been reported previously in abstract form (20, 21, 24) 82

MATERIALS AND METHODS 83

Antimicrobial test agents. Avibactam was supplied by Novexel (Romainville, France). 84

Ceftazidime pentahydrate (Fortum®) was supplied by Sandoz. Amoxicillin 1g- 85

clavulanate 0.2 g, (Augmentin®) was supplied by Glaxo Smithkline. Piperacillin 4 g – 86


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tazobactam 0.5 g (Tazocillin®) was supplied by Wyeth-Lederle. Cefotaxime, piperacillin, 87

and tazobactam were purchased from Sigma Aldrich (France). 88

Bacterial isolates. Twenty different isolates of Enterobacteriaceae, 5 ceftazidime-89

susceptible and 15 ceftazidime-resistant isolates with different β-lactamases were tested 90

in this study. Seventeen clinical isolates were obtained from different French hospitals 91

including the CTX-M E. coli which were provided by Guillaume Arlet (Hôpital Tenon, 92

Paris, France) . Three CTX-M K. pneumoniae isolates were kindly provided by Robert 93

Bonomo (Case Western Reserve University School of Medicine, Cleveland, Ohio, USA). 94

The β-lactamases produced by these strains were well characterized and are listed in 95

the tables (1a; 1b). 96

Determination of β-lactamases. Detection and characterization of β-lactamases 97

expressed in various bacterial strains was achieved by bla gene detection, eventually 98

combined with isoelectric focusing (IEF) of cell extracts. 99

Gene detection. Bacterial DNA was submitted to PCR amplification profiling using the 100

appropriate primers for detection of the most common bla gene families (28–30): 101

Class A: TEM, SHV, VEB, PER, GES, CARB, CTX-M, and KPC families 102

Class B: IMP and VIM families 103

Class C: CMY-1/MOX, CMY-2, DHA, ACC, ACT-1, and FOX plasmidic 104

subgroups 105

Class D: OXA-1, OXA-2, OXA-10/13, and, OXA-18/45 families 106


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The PCRs were performed using the Ready-To-Go reagents (GE Healthcare) according 107

to the manufacturer’s instructions. Briefly, cell lysis was performed at 99°C for 3 min, 108

followed by 30 cycles of amplification (94°C for 1 min, 55°C for 1 min, 72°C for 1 min), 109

and a final extension at 72°C for 5 min. 110

In vitro susceptibility. Minimum inhibitory concentration (MIC) determinations were 111

performed using the guidelines of the Clinical and Laboratory Standards Institute (CLSI) 112

for antimicrobial susceptibility testing with cation-adjusted Mueller-Hinton broth (31). 113

The MIC was defined as the lowest concentration that inhibited visual growth. MICs for 114

ceftazidime, piperacillin, or cefotaxime were determined with (i) avibactam or 115

tazobactam at ratios of 4:1 and 8:1 and (ii) avibactam or tazobactam used at a fixed 4 116

µg/ml with variable concentrations of either ceftazidime, piperacillin, or cefotaxime. The 117

interpretive criteria considered for ceftazidime and cefotaxime in combination with 4 118

µg/ml avibactam were the ones defined by CLSI for the antibiotics alone (32). All other 119

combinations employed a fixed ratio of antibiotic-inhibitor to help interpret the fixed ratio 120

in vivo data rather than to determine sensitivity/resistance. 121

Mice. Male 5- to 6-week-old (20 – 23 g) male ICR (CD-1®) mice (Charles River 122

Laboratories, France) were used in the acute lethal septicemia model. Mice were 123

housed in groups of 5 to10 with free access to food and water in the Microbiology in vivo 124

Laboratory (Antiinfective Research, Lavoisier Building, Novexel, Romainville, France). 125

Experiments were carried out according to protocols approved by the Institutional 126

Animal Care and Ethical Committee (Novexel, Romainville, France) and authorization 127

from the Département de Santé Véterinaire, Perfecture de Bobigny, France. 128


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Murine acute lethal septicemia. Mice were infected by intraperitoneal injection of the 129

bacterial strains in 5% hog gastric mucin (Sigma) containing inocula of 1.5 x 108 to 2.7 130

x109 CFU in 0.5 ml volume. Groups of 10 mice were dosed subcutaneously with 131

antibiotic or antibiotic-inhibitor combination at different doses (dose ranges of 1.5 mg/kg 132

to 100 mg/kg) (1 dose per group) in 0.2 ml saline. Dosing was performed twice at 1- and 133

4- hours post-infection. A control group of 10 to 15 infected mice received only saline at 134

the dosing times. 135

In this model, infected mice developed septicemia and became moribund within 48 136

hours of infection unless they received adequate therapy. Efficacy was monitored using 137

survival as the end point, with observation continued for five days post-treatment. 138

Animals under test were inspected multiple times/day and stressed animals were 139

euthanized. 140

The 50% effective dose (ED50

) is reported as unit dose of the antibiotic component in 141

mg/kg. As two doses for each dosage group (1 and 4 hours post-infection) were utilized, 142

the total ED50

should be interpreted as ED50

x 2 mg/kg. For the antibiotic-inhibitor 143

treatments, the dose reported is the unit dose of the antibiotic. Thus, a 4:1 ceftazidime-144

avibactam combination ED50 of 10 mg/kg represents 10 mg/kg ceftazidime + 2.5 mg/kg 145

avibactam. The ED50

values were calculated by log-probit analysis (33) using software 146

written in house. 147

RESULTS AND DISCUSSION 148

In vitro susceptibility. Tables 1(a) and 1(b) show the MICs for ceftazidime, amoxicillin, 149

cefotaxime and piperacillin either alone or in combination with avibactam or tazobactam 150


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against ceftazidime -susceptible and –resistant isolates of Enterobacteriaceae. Using 151

the CLSI-approved method of reporting the MIC of ceftazidime and cefotaxime, in the 152

presence of a fixed 4 µg/ml of avibactam, the activity of both ceftazidime and cefotaxime 153

were restored against the class A and class C β-lactamase-producing isolates. 154

MICs were also determined using fixed antibiotic-inhibitor ratios, allowing a direct 155

comparison with the in vivo data in Tables 2 and 3. Avibactam at both 4:1 and 8:1 ratios 156

significantly improved the in vitro activity of ceftazidime against both class A- and class 157

C-producing isolates. Avibactam when combined with cefotaxime at a 4:1 ratio also 158

significantly improved the activity of the antibiotic against AmpC- and CTX-M-producing 159

E. coli and K. pneumoniae isolates, while piperacillin-tazobactam at a 4:1 ratio was not 160

active against the AmpC-producing isolates (Table 1b). 161

Saline-treated control animals. Mice infected with the ceftazidime-susceptible and -162

resistant Enterobacteriaceae isolates and treated with saline post-infection succumbed 163

to the infection within 48 hours (100% of animals), thereby demonstrating the 164

pathogenicity of the isolates used in the study. 165

Studies with ceftazidime-avibactam at ratios of 4:1 and 8:1 (Table 2). In initial 166

studies, the optimal ratio of antibiotic-inhibitor for in vivo efficacy against the ceftazidime-167

susceptible and resistant isolates was investigated. Two different ratios of 4:1 and 8:1 168

of ceftazidime-avibactam were tested against 14 different Enterobacteriaceae isolates, 169

comprising five ceftazidime-susceptible isolates (2 Escherichia coli, 2 Klebsiella 170

pneumoniae, 1 Providencia stuartii) and nine ceftazidime-resistant isolates (1 E. coli, 3 171

K. pneumoniae, 3 Enterobacter cloacae, 2 Citrobacter freundii). Amoxicillin 1g – 172


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clavulanic acid 0.2 g and piperacillin 4 g – tazobactam 0.5 g were used as reference 173

comparator agents against the ESBL- and AmpC-producing organisms. 174

Against ceftazidime-susceptible strains of K. pneumoniae, E. coli, and P. stuartii, 175

ceftazidime alone, and in combination with avibactam, demonstrated better efficacy than 176

the control agents, with unit dose ED50

of <1.5 to 9 mg/kg compared to 12 to >50 mg/kg 177

for amoxicillin-clavulanate and >50 mg/kg for piperacillin-tazobactam. Furthermore, the 178

efficacy of ceftazidime against susceptible isolates was not compromised by combining 179

it with avibactam. Against class A (TEM, SHV) β-lactamase-producing strains of K. 180

pneumoniae and E. coli, addition of avibactam restored ceftazidime efficacy (unit dose 181

ED50

<5 – 29 mg/kg), in particular, against SHV-producing isolates where cetazidime 182

alone, amoxicillin-clavulanate, and piperacillin-tazobactam were less active (ED50 unit 183

dose of: >90 mg/kg; 20 - >90 mg/kg; 39 - >90 mg/kg, respectively). 184

While ceftazidime, piperacillin-tazobactam and amoxicillin-clavulanate were inactive 185

against the class C cephalosporinase-producing species of E. cloacae and C. freundii, 186

the ED50 values for the ceftazidime-avibactam combinations were consistently lower 187

than those of ceftazidime alone, with the 4:1 and 8:1 ratios being equally active. 188

Studies on ceftazidime-avibactam at a ratio of 4:1 (Table 3). Based on the initial 189

studies, additional septicemia studies were performed with 4:1 ceftazidime-avibactam 190

against eight ceftazidime-resistant isolates producing AmpC (2 isolates) and/or CTX-M 191

ESBLs (6 isolates, one of them also carrying the ESBL gene blaSHV-5). Their β-lactamase 192

profiles are shown in Table 3. The comparators used throughout these studies were 193

piperacillin and piperacillin-tazobactam. When a CTX-M producing isolate was under 194


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test, cefotaxime was an additional comparator, both alone and combined with 195

avibactam. 196

Against the two AmpC-producing isolates, ceftazidime-avibactam was superior to 197

piperacillin-tazobactam both in vitro (Table 1b) and in vivo (Table 3). 198

Against the CTX-M-producing isolates of E. coli and K. pneumoniae, tazobactam 199

afforded no protection to piperacillin in vivo. In vitro, three strains showed piperacillin 200

MICs of 2-16 µg/ml in the presence of tazobactam against CTX-M producers. 201

Avibactam, is an efficient inhibitor of these enzymes and protected both ceftazidime and 202

cefotaxime in vitro, although in vivo only the ceftazidime combination was broadly 203

effective against these strains (ED50

unit doses 2 - 27 mg/kg). However against K. 204

pneumoniae strain 456, ED50 values were essentially identical for ceftazidime-avibactam 205

and cefotaxime-avibactam (14 and 18 mg/kg respectively). 206

Septicemia studies were initially performed on 14 different Enterobacteriaceae isolates, 207

a few susceptible to ceftazidime but most ceftazidime-resistant due to production of β-208

lactamases. In these pre-clinical studies, the septicemia model did not show any 209

significant differences between the 4:1 and 8:1 ratios of ceftazidime-avibactam. 210

Ultimately, a 4:1 ratio of ceftazidime-avibactam was selected for clinical development 211

based on a number of factors, including in vitro, in vivo, and the hollow-fiber infection 212

model data (8, 9, 12, 13, 20, 21, 23-26, 34). 213

Eventually, eight isolates (two AmpC producers and six CTX-M producers) were studied 214

at a 4:1 ratio of ceftazidime-avibactam and compared to piperacillin-tazobactam and 215

cefotaxime-avibactam respectively. The in vivo efficacy of ceftazidime-avibactam 216


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combinations was consistently better than other antibiotic-β-lactamase inhibitor 217

combinations, such as piperacillin-tazobactam. Since tazobactam is a poor inhibitor of 218

both these enzymes (35-37), it is not surprising that the piperacillin-tazobactam 219

combination was uniformly less active, whereas the ceftazidime-avibactam combination 220

afforded protection in the septicemia model. Furthermore, unlike clavulanic acid, 221

avibactam did not induce the AmpC β-lactamase in three strains of E. cloacae (38). 222

Cefotaxime and cefotaxime-avibactam were used as comparators in studies on CTX-M-223

producing isolates. Although avibactam generally protected cefotaxime in these studies 224

when dosed twice at 1 hour and 4 hours post-infection, protection was poor against 225

some CTX-M producers, particularly E. coli E4. Dosing three times also failed to give a 226

measurable ED50 against this isolate (dosing at 1, 4 and 7 hours post-infection), both 227

cefotaxime and cefotaxime-avibactam 4:1 ratio having an ED50 unit dose of >90 mg/kg 228

(data not shown). 229

CONCLUSION 230

These data show that a 4:1 ratio of ceftazidime-avibactam proved effective against a 231

range of Enterobacteriaceae in a mouse septicemia model where ceftazidime alone was 232

ineffective due to production of β-lactamase by the infecting organism. Avibactam was 233

also tested in combination with a number of other β-lactams, but none of these 234

combinations had potency equivalent to ceftazidime-avibactam. The KPC β-lactamases 235

are an important cause of cephalosporin resistance and no KPCs were included in this 236

study. However, Endimiani et al have demonstrated in vivo the efficacy of ceftazidime-237


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avibactam at a ratio of 4:1against two Enterobacteriaceae strains producing KPC β-238

lactamases (22). 239

Ceftazidime-avibactam has successfully completed two phase 2 clinical studies (39, 40) 240

and is currently in phase 3 clinical development for treatment of complicated intra-241

abdominal infection, urinary tract infection, nosocomial pneumonia, and infections with 242

ceftazidime-resistant pathogens (http://clinicaltrials.gov). Based on both pre-clinical and 243

phase 2 data, ceftazidime-avibactam seems to be a promising treatment option against 244

the widespread multi-drug resistant Enterobacteriaceae and P. aeruginosa isolates 245

which currently pose a worldwide problem (41, 42). 246

ACKNOWLEDGEMENTS 247

We thank R. Bonomo and G. Arlet for supplying some of the isolates used in this study. 248

This study was funded by Novexel. Ceftazidime-avibactam is now being developed by 249

AstraZeneca and Forest-Cerexa. PL, AMG, CM, KC received compensation fees for 250

services in relation to preparing the manuscript, which was funded by AstraZeneca. PL, 251

AMG, LL, CM, JP, KC are ex-employees of Novexel. 252


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404


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Table 1(a). MICs against isolates of Enterobacteriaceae reported in Table 2 405

406

Isolate Phenotype β-lactamase

MIC (µg/ml)

Ceftazidime

Amoxicillin-clavulanate4

Piperacillin-tazobactam5

Alone

+ Avibactam

4 µg/ml1

4:12 8:13

E. coli 250GR12

CAZ-S 0.06 0.06 0.06 0.06 2 2

E. coli 250GR43

CAZ-S 0.25 0.12 0.12 0.12 4 1

K. pneumoniae

283GR4

CAZ-S 0.25 0.5 0.25 0.25 16 32

K. pneumoniae

283IP53

CAZ-S <0.12 <0.12 0.12 0.12 4 4

P. stuartti 321UC1

CAZ-S 1 0.5 0.5 1 64 4


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407

E. coli 250BE1

CAZ-R SHV-4 >128 0.25 2 4 8 16

K. pneumoniae

283IP10

CAZ-R AmpC; SHV-4 64 0.5 1 2 8 >32

K. pneumoniae

283IP35

CAZ-R SHV-2 >128 0.25 0.5 1 64 32

K. pneumoniae

283IP84

CAZ-R TEM-3 64 0.25 2 8 32 32

E. cloacae 293GR8

CAZ-R AmpC >128 0.5 1 2 >128 32

E. cloacae 293GR38

CAZ-R AmpC 64 0.5 1 1 64 >32

E. cloacae P99

CAZ-R AmpC >128 0.5 0.5 1 >128 >32

C. freundii 261GR3

CAZ-R AmpC >128 0.06 1 1 >128 32

C. freundii 261GR6

CAZ-R AmpC >128 0.5 2 2 >128 32


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1 : MICs for ceftazidime in the presence of a fixed 4 µg/ml avibactam 408

2 : MICs for ceftazidime when combined with avibactam in a 4:1 weight/weight ratio 409

3 : MICs for ceftazidime when combined with avibactam in a 8:1 weight/weight ratio 410

4: Commercially available amoxicillin-clavulanate (Augmentin®: amoxicillin 1 g – clavulanate 0.2 g) 411

5: Commercially available piperacillin-tazobactam (Tazocillin®: piperacillin 4 g – tazobactam 0.5 g) 412


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413

Table 1(b). MICs against isolates of Enterobacteriaceae reported in Table 3 414

Isolate β-lactamase

MIC (µg/ml)1

Ceftazidime

Cefotaxime

Piperacillin

Alone

+ Avibactam

Alone

+Avibactam

Alone

+ Tazobactam

4 µg/ml

4:1

4 µg/ml

4:1

4 µg/ml

4:1

K. pneumoniae 283IP10

AmpC; SHV-4 >128 0.5 1 >128 0.25 NT >128 64 >128

E. cloacae P99

AmpC >128 0.5 0.5 >128 0.25 NT >128 >128 >128

E. coli TN06

CTX-M-2; TEM-1 >128 0.5 4 >128 0.125 2 >128 16 16

E. coli E4

CTX-M-16; TEM-1 >128 1 4 >128 0.5 1 >128 8 8

E. coli TN03

CTX-M-15; TEM-1; OXA-1

>128 0.25 2 >128 <0.125 0.5 >128 2 2

K. pneumoniae 465

CTX-M-2; TEM-1 64 2 4 >128 0.25 4 >128 >128 >128

K. pneumoniae 253

CTX-M-2; SHV-5; TEM-2

>128 2 8 >128 0.25 4 >128 >128 >128

K. pneumoniae K4


>128 1 4 >128 <0.125 4 >128 >128 >128

415


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1: MICs for ceftazidime, cefotaxime, or piperacillin were determined with avibactam or tazobactam at a weight/weight ratio of 4:1 and 416

avibactam or tazobactam used at a fixed 4µg/ml concentration. 417

NT: Not tested 418


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419

Table 2. Efficacy of ceftazidime-avibactam against ESBL and AmpC producing strains of Enterobacteriaceae 420 421

Isolate

Phenotype

β-lactamase

MIC (µg/ml)

ED50

1 mg/kg (95% Confidence Limits)

Ceftazidime

Augmentin1 Tazocillin2

Ceftazidime

Alone

+ Avibactam

4:1

8:1

E. coli 250GR12

CAZ-S

0.06

<5

<5

<5

12 (5-25)

>50

E. coli 250GR43

CAZ-S

0.25

9 (3-17)

5 (1-8)

6 (4-11)

22 (14-36)

>50

K. pneumoniae 283GR4

CAZ-S

0.25

<1.5

<1.5

<1.5

>50

>50


CAZ-S

<0.12

4 (0-5)

4.5 (0-5)

4.5 (0-5)

27 (0-107)

>50

P. stuartti 321UC1

CAZ-S

1

6 (1-14)

5 (1-10)

4 (1-7)

>50

>50

E. coli 250BE1

CAZ-R

SHV-4

>128

>90

16 (10-27)

16 (10-27)

49 (28-533)

39 (10-192)


CAZ-R

AmpC; SHV-4

64

>90

5 (1-8)

9 (6-13)

20 (12-34)

>90


CAZ-R

SHV-2

>128

>90

29 (19-50)

18 (5-33)

>90

>90


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CAZ-R

TEM-3

64

>90

<5

<5

43 (18-134)

>90

E. cloacae 293GR8

CAZ-R

AmpC

>128

>90

<10

11 (4-19)

>90

>90

E. cloacae 293GR38

CAZ-R

AmpC

64

>90

58 (38-93)

65 (41-126)

>90

>90

E. cloacae P99

CAZ-R

AmpC

>128

85 (49-75)

5 (3-9)

10 (0-10)

>90

43 (7-75)

C. freundii 261GR3

CAZ-R

AmpC

>128

>90

13 (11-15)

14 (11-26)

>90

>90

C. freundii 261GR6

CAZ-R

AmpC

>128

84 (61-145)

9 (0-10)

9 (0-10)

>90

>90

422 1: Mice were dosed twice at 1- and 4- hours post-infection; Unit dose ED50 of the antibiotic component is reported here 423

2: Commercially available amoxicillin-clavulanate (Augmentin®: amoxicillin 1 g – clavulanate 0.2 g) 424

3: Commercially available piperacillin-tazobactam (Tazocillin®: piperacillin 4 g – tazobactam 0.5 g) 425


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Table 3. Efficacy of ceftazidime-avibactam against AmpC and CTX-M producing strains of Enterobacteriaceae 426

427

Isolate β-lactamase MIC (µg/ml)

ED50

1 mg/kg (95% confidence limit)

Ceftazidime

Piperacillin

Cefotaxime

Ceftazidime Alone + Avibactam

Alone+ Tazobactam

Alone+ Avibactam

4:1 4:1 4:1

K. pneumoniae 283IP10 AmpC; SHV-4 >128

>90

7 (0-10)

>90

>90

NT

NT

E. cloacae P99 AmpC >128 85 (49-75) 5 (3-9)

>90

>90

NT

NT

E. coli TN06 CTX-M-2; TEM-1 >128

>90

21 (16-31)

>90

>90

>90

55 (44-70)

E. coli E4 CTX-M-16; TEM-1 >128 74 (60->500)

13 (9-29)

>90

>90

>90

>90

E. coli TN03 CTX-M-15; TEM-1; OXA-1

>128

>90

2 (1-4)

>90

>90

NT

NT

K. pneumoniae 465

CTX-M-2; TEM-1B 64

>90

18 (6-32)

>90

>90

>90

14 (6-61)

K. pneumoniae 253

CTX-M-2; SHV-5; TEM-2

>128 127 (82-

>500) 27 (14-53)

>90

>90

165 (94-

>500) 70 (42->155)

K. pneumoniae K4


>128

>90

21 (12-40)

>90

>90

NT

NT

428

1: Mice were dosed twice at 1- and 4- hours post-infection; Unit dose ED50 of the antibiotic component is reported here 429


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NT: not tested 430


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